Psychopharmacology (1992) 108:333 337
Psychopharmacology © Springer-Verlag 1992
Are there changes in sensitivity to 5-HT3 receptor ligands following chronic diazepam treatment? N i c k A n d r e w s and S a n d r a E. File
Psychopharmacology Research Unit, UMDS Division of Pharmacology, Guy's Hospital, London SE1 9RT, UK Received October 11, 1991 / Final version February 20, 1992 Administration of 1-(3-chlorophenyl)-biguanide (mCPB), a 5-HT3 receptor agonist (1 and 10 mg/kg IP), was found to be significantly anxiogenic in vehicle treated rats tested in the plus-maze, while having no significant effect in rats withdrawn for 24 h from 21 days diazepam treatment (2 mg/kg/day), suggesting a decreased agonist action at 5-HT3 receptors following withdrawal from chronic diazepam treatment. In the social interaction test, diazepam withdrawn rats showed a significant decrease in social interaction when compared to the chronic vehicle treated group. This anxiogenic response was reversed by low doses of zacopride (0.0001-0.01 mg/kg IP); in the vehicle treated animals 0.1 mg/kg was significantly anxiogenic. The overall pattern of results with zacopride is explained by suggesting that the anxiogenic effects of high doses of zacopride are detectable at low levels of 5-HT function and are due to an agonist action of the S-isomer in the rat at 5-t-IT3 receptors. The anxiolytic action of low doses is attributed to the R-isomer acting at the R-zacopride binding site and is enhanced in conditions of high 5-HT function, e.g. in the diazepam withdrawn rats. If this hypothesis is correct, then we would predict the R-isomer alone would be more effective in reversing the anxiogenic effects of diazepam withdrawal than the racemate, used here. Abstract.
Benzodiazepine Zacopride
K e y words:
5-HT3
Dependence - A n x i e t y -
Anxiety is one of the major symptoms experienced by patients following discontinuation of a period of chronic benzodiazepine administration, with approximately 15 % of patients administered benzodiazepines for more than 6-8 weeks experiencing some difficulty following termination (Dunbar et al. 1989). It has also been possible to detect changes in animal tests of anxiety, as well as weight loss and a decreased seizure threshold following Offprint requests to:
S.E. File
chronic benzodiazepine treatment in rodents (File 1990). Weight loss is not often reported in patients, and of the behavioural changes that can be detected in rats, those indicating increased anxiety are certainly the most clinically relevant. Our recent studies have indicated that there may be important changes in the 5-HT system after a period of chronic diazepam treatment. Increased 5-HT release has been found in hippocampal slices taken 24 h to 2 weeks after the last dose of diazepam, from rats treated for 21 days with low daily doses (Hitchcott et al. 1990). The increased hippocampal 5-HT release was accompanied by an increase in 45Caa+ uptake into hippocampal synaptosomes and both of these changes were reversed by baclofen at concentrations not affecting tissue taken from control-treated animals (Andrews et al. 1992). This enhanced sensitivity to the effects of baclofen after a period of chronic diazepam treatment was also found behaviourally. Low doses of baclofen reversed the anxiogenic responses detected in the social interaction test after withdrawal from diazepam, and these doses were ineffective in control-treated animals (File et al. 1991). Similar changes in the sensitivity of the 5-HT system, were indicated by the enhanced anxiolytic efficacy of very low doses of buspirone in diazepam withdrawn rats (File and Andrews 1991). Higher doses of 5-HTIA receptor agonists exacerbated symptoms of benzodiazepine withdrawal (File and Andrews 1991; Goudie and Leathley 1991). The purpose of the present investigation was to determine whether there was a changed sensitivity of 5t-IT 3 receptors after a period of chronic diazepam treatment. The effects of the 5 HT 3 receptor antagonist, zacopride (Smith et al. 1988 and Barnes et al. 1988) and the 5-HT3 receptor agonist, 1-(3-chloro-phenyl)-biguanide (mCPB, Kilpatrick et al. 1990) were therefore compared in control-treated rats and in those withdrawn from 21 days of diazepam treatment (2 mg/kg/day). At the time that these experiments were conducted, zacopride had not been found to have anxiolytic actions in the elevated plus-maze test (File and Johnston 1989),
334 a l t h o u g h there was a report o f anxiolytic action in the social i n t e r a c t i o n a n d the black-white crossing tests (Costall et al. 1988). There has s u b s e q u e n t l y b e e n a report o f anxiolytic actions in the elevated p l u s - m a z e ( D u n n et al. 1991). The effects o f zacopride o n the a n x i o g e n i c response to b e n z o d i a z e p i n e w i t h d r a w a l has n o t yet been reported. However, Costall et al. (1989) reported t h a t in the social i n t e r a c t i o n test the 5 - H T 3 receptor a n t a g o n i s t , o n d a n s e t r o n , was able to reverse the a n x i o g e n i c effects of w i t h d r a w a l f r o m a fairly high dose o f d i a z e p a m (10 m g / k g orally twice daily). This reversal a p p e a r e d to be a f u n c t i o n a l a n t a g o n i s m (i.e., simply a m a t t e r o f the additive effects o f a n anxiolytic a n d a n a n x i o g e n i c action), since the doses that were effective in w i t h d r a w a l were also a n x i o l y t i c in controls. I f the i n t e r a c t i o n between the 5 - H T 3 receptor ligands a n d b e n z o d i a z e p i n e w i t h d r a w a l is a f u n c t i o n a l one t h e n zacopride s h o u l d reduce w i t h d r a w a l o n l y at doses t h a t are anxiotytic in c o n t r o l s animals, a n d m C P B s h o u l d e n h a n c e w i t h d r a w a l at a n x i o g e n i c doses. T h e social i n t e r a c t i o n test was selected for the assessm e n t o f zacopride's effects, since it was t h o u g h t to be the test m o s t likely to be sensitive to zacopride. T h e behavioural effects o f m C P B have n o t yet been published, a n d because o f a limited supply o f the drug, the elevated plus-maze was selected for e x p e r i m e n t 2 since this uses only h a l f the n u m b e r o f rats needed for social i n t e r a c t i o n , a n d in a d d i t i o n measures o f l o c o m o t o r activity a n d rearing were taken. The doses o f zacopride were based o n those previously f o u n d to be active in a n i m a l tests of anxiety (Costall et al. 1988 ; Barnes et al. 1990) a n d those o f m C P B were chosen o n the basis o f the relative efficacies o f m C P B a n d 5 - H T 3 a n t a g o n i s t s in vitro ( K i l p a t r i c k et al. 1990).
Materials and methods Animals. For all experiments male hooded Lister rats weighing approximately 150 g at the start of chronic treatment were housed in groups of five with food and water freely available. Lights were on from 0600 to 1800 hours. Five days before testing, animals allocated to experiment 1 were singly housed and allocated test partners on the basis of body weight (partners did not differ by more than 5 g). Both partners received the same chronic treatment and also the same acute treatment on the test day. In experiment 2 the animals tested in the plus-maze were left in their home cages in groups of five up to and including the test day. Drugs. Diazepam (1 mg/ml) (courtesy of Roche Products Ltd) was suspended in distilled water containing 1 drop of Tween 20 per 20 ml water, and sonicated in an ultrasonic water bath for 30 minutes prior to intraperitoneal administration. Rats allocated to chronic diazepam treatment received daily injections of 2 mg/kg. The vehicle treated animals received 2 ml/kg distilled water containing i drop of Tween 20 per 20 ml water. Zacopride (the racemic mixture, courtesy of Delalande, France) and 1-(3-chloro-phenyl)biguanide; mCPB (courtesy of MSD, UK) were dissolved in distilled water and administered intraperitoneally 30 and 15 min, respectively, prior to testing. Rats allocated to the acute control treatment groups received distilled water injections at the appropriate times before test. Apparatus. The social interaction test arena was a wooden box 60 x 60 cm with 35 cm high walls and was lit by dim light (35
radiometric lux). A camera was mounted vertically above the arena and the rats were observed from a video monitor in the adjacent room. Infra-red photocells were located in the walls of the box; those 4.5 cm above the floor recorded locomotor activity and those 12.5 cm above the floor recorded rearing of the animals. The output from both sets of photocells and the scores of the observer were entered into a computer for later analysis (see File 1980 for more details of this test). The plus-maze was made of wood and consisted of two opposite open arms - 50 cm x 10 cm and two opposite closed arms 50 x 10 x 40 cm with an open roof. The arms were connected by a central square 10 x 10 cm, thus the maze formed a "plus" shape. The maze was elevated 50 cm from the floor.
Procedure. In experiment 1,200 animals were randomly allocated between the two chronic treatment groups, such that 100 rats received an IP injection of vehicle on each of 21 consecutive days, and 100 rats received 21 daily injections of 2 mg/kg IP diazepam. On day 20 the rats were familiarised with the social interaction arena in the undrugged state, with their partners, for 4.5 min and then injected according to their normal chronic treatment. The following day the animals were placed alone in the box (again in the undrugged state) for 7.5 rain, and injected immediately after removal from the box. This was in order to test all animals in the low light, familiar test condition, which is the most sensitive to anxiogenic effects. Twenty-four hours after the 21 st injection, animals of both chronic treatment groups were randomly assigned to either control or zacopride (0.1, 0.01, 0,001 or 0.0001 mg/kg) groups and tested for 4.5 min in social interaction. The time spent in active social interaction was scored by an observer blind to the drug treatment. At the end of each trial the arena was thoroughly wiped. In experiment 2, 44 altimals were treated for 21 days with either vehicle or diazepam and on day 22 were injected with vehicle, 1 or 10 mg/kg mCPB 15 min prior to being placed singly in the same arena as was used for the social interaction test. This was in order to obtain automated measures of locomotor activity and rearing. After 5 min the animals were immediately placed in the plus-maze facing a closed arm and observed for a further 5 rain. During this time the number of open and closed arm entries and the time spent on the open and closed arms were recorded and entered into an IBM computer for later analysis. Statistics. Data were initially analysed by two factor analysis of variance with chronic treatment as one factor and acute treatment as the other factor. A significant acute x chronic treatment interaction would indicate that acute drug treatment was producing different effects depending on whether the animals had previously received chronic vehicle or diazepam. Post-hoc Duncan tests were used for comparisons of the effects of individual doses with the relevant control group.
Results It c a n be seen f r o m Fig. 1 that the rats w i t h d r a w n f r o m d i a z e p a m a n d given n o acute d r u g t r e a t m e n t h a d significantly lower social i n t e r a c t i o n scores t h a n the c o n t r o l treated animals, i n d i c a t i n g a n a n x i o g e n i c w i t h d r a w a l response. There was a significant c h r o n i c x acute treatm e n t i n t e r a c t i o n [F(4,88)= 2.5, P = 0.05], i n d i c a t i n g that there was a different response to zacopride d e p e n d i n g o n the previous c h r o n i c t r e a t m e n t . I n the c o n t r o l - t r e a t e d rats, zacopride tended to reduce the time spent in social i n t e r a c t i o n , whereas in the d i a z e p a m - w i t h d r a w n rats the trend was to increase social i n t e r a c t i o n , see Fig. 1. O n post-hoc tests, the reversal o f the a n x i o g e n i c w i t h d r a w a l response reached significance for rats treated with zacopride (0.001 mg/kg), a n d the a n x i o g e n i c effect in c o n t r o l -
335 140-
120-
DIAZEPAM WITHDRAWAL
CONTROL
(0
u~ 1 2 0 Z
o
~S
Psychopharmacology © Springer-Verlag 1992
Are there changes in sensitivity to 5-HT3 receptor ligands following chronic diazepam treatment? N i c k A n d r e w s and S a n d r a E. File
Psychopharmacology Research Unit, UMDS Division of Pharmacology, Guy's Hospital, London SE1 9RT, UK Received October 11, 1991 / Final version February 20, 1992 Administration of 1-(3-chlorophenyl)-biguanide (mCPB), a 5-HT3 receptor agonist (1 and 10 mg/kg IP), was found to be significantly anxiogenic in vehicle treated rats tested in the plus-maze, while having no significant effect in rats withdrawn for 24 h from 21 days diazepam treatment (2 mg/kg/day), suggesting a decreased agonist action at 5-HT3 receptors following withdrawal from chronic diazepam treatment. In the social interaction test, diazepam withdrawn rats showed a significant decrease in social interaction when compared to the chronic vehicle treated group. This anxiogenic response was reversed by low doses of zacopride (0.0001-0.01 mg/kg IP); in the vehicle treated animals 0.1 mg/kg was significantly anxiogenic. The overall pattern of results with zacopride is explained by suggesting that the anxiogenic effects of high doses of zacopride are detectable at low levels of 5-HT function and are due to an agonist action of the S-isomer in the rat at 5-t-IT3 receptors. The anxiolytic action of low doses is attributed to the R-isomer acting at the R-zacopride binding site and is enhanced in conditions of high 5-HT function, e.g. in the diazepam withdrawn rats. If this hypothesis is correct, then we would predict the R-isomer alone would be more effective in reversing the anxiogenic effects of diazepam withdrawal than the racemate, used here. Abstract.
Benzodiazepine Zacopride
K e y words:
5-HT3
Dependence - A n x i e t y -
Anxiety is one of the major symptoms experienced by patients following discontinuation of a period of chronic benzodiazepine administration, with approximately 15 % of patients administered benzodiazepines for more than 6-8 weeks experiencing some difficulty following termination (Dunbar et al. 1989). It has also been possible to detect changes in animal tests of anxiety, as well as weight loss and a decreased seizure threshold following Offprint requests to:
S.E. File
chronic benzodiazepine treatment in rodents (File 1990). Weight loss is not often reported in patients, and of the behavioural changes that can be detected in rats, those indicating increased anxiety are certainly the most clinically relevant. Our recent studies have indicated that there may be important changes in the 5-HT system after a period of chronic diazepam treatment. Increased 5-HT release has been found in hippocampal slices taken 24 h to 2 weeks after the last dose of diazepam, from rats treated for 21 days with low daily doses (Hitchcott et al. 1990). The increased hippocampal 5-HT release was accompanied by an increase in 45Caa+ uptake into hippocampal synaptosomes and both of these changes were reversed by baclofen at concentrations not affecting tissue taken from control-treated animals (Andrews et al. 1992). This enhanced sensitivity to the effects of baclofen after a period of chronic diazepam treatment was also found behaviourally. Low doses of baclofen reversed the anxiogenic responses detected in the social interaction test after withdrawal from diazepam, and these doses were ineffective in control-treated animals (File et al. 1991). Similar changes in the sensitivity of the 5-HT system, were indicated by the enhanced anxiolytic efficacy of very low doses of buspirone in diazepam withdrawn rats (File and Andrews 1991). Higher doses of 5-HTIA receptor agonists exacerbated symptoms of benzodiazepine withdrawal (File and Andrews 1991; Goudie and Leathley 1991). The purpose of the present investigation was to determine whether there was a changed sensitivity of 5t-IT 3 receptors after a period of chronic diazepam treatment. The effects of the 5 HT 3 receptor antagonist, zacopride (Smith et al. 1988 and Barnes et al. 1988) and the 5-HT3 receptor agonist, 1-(3-chloro-phenyl)-biguanide (mCPB, Kilpatrick et al. 1990) were therefore compared in control-treated rats and in those withdrawn from 21 days of diazepam treatment (2 mg/kg/day). At the time that these experiments were conducted, zacopride had not been found to have anxiolytic actions in the elevated plus-maze test (File and Johnston 1989),
334 a l t h o u g h there was a report o f anxiolytic action in the social i n t e r a c t i o n a n d the black-white crossing tests (Costall et al. 1988). There has s u b s e q u e n t l y b e e n a report o f anxiolytic actions in the elevated p l u s - m a z e ( D u n n et al. 1991). The effects o f zacopride o n the a n x i o g e n i c response to b e n z o d i a z e p i n e w i t h d r a w a l has n o t yet been reported. However, Costall et al. (1989) reported t h a t in the social i n t e r a c t i o n test the 5 - H T 3 receptor a n t a g o n i s t , o n d a n s e t r o n , was able to reverse the a n x i o g e n i c effects of w i t h d r a w a l f r o m a fairly high dose o f d i a z e p a m (10 m g / k g orally twice daily). This reversal a p p e a r e d to be a f u n c t i o n a l a n t a g o n i s m (i.e., simply a m a t t e r o f the additive effects o f a n anxiolytic a n d a n a n x i o g e n i c action), since the doses that were effective in w i t h d r a w a l were also a n x i o l y t i c in controls. I f the i n t e r a c t i o n between the 5 - H T 3 receptor ligands a n d b e n z o d i a z e p i n e w i t h d r a w a l is a f u n c t i o n a l one t h e n zacopride s h o u l d reduce w i t h d r a w a l o n l y at doses t h a t are anxiotytic in c o n t r o l s animals, a n d m C P B s h o u l d e n h a n c e w i t h d r a w a l at a n x i o g e n i c doses. T h e social i n t e r a c t i o n test was selected for the assessm e n t o f zacopride's effects, since it was t h o u g h t to be the test m o s t likely to be sensitive to zacopride. T h e behavioural effects o f m C P B have n o t yet been published, a n d because o f a limited supply o f the drug, the elevated plus-maze was selected for e x p e r i m e n t 2 since this uses only h a l f the n u m b e r o f rats needed for social i n t e r a c t i o n , a n d in a d d i t i o n measures o f l o c o m o t o r activity a n d rearing were taken. The doses o f zacopride were based o n those previously f o u n d to be active in a n i m a l tests of anxiety (Costall et al. 1988 ; Barnes et al. 1990) a n d those o f m C P B were chosen o n the basis o f the relative efficacies o f m C P B a n d 5 - H T 3 a n t a g o n i s t s in vitro ( K i l p a t r i c k et al. 1990).
Materials and methods Animals. For all experiments male hooded Lister rats weighing approximately 150 g at the start of chronic treatment were housed in groups of five with food and water freely available. Lights were on from 0600 to 1800 hours. Five days before testing, animals allocated to experiment 1 were singly housed and allocated test partners on the basis of body weight (partners did not differ by more than 5 g). Both partners received the same chronic treatment and also the same acute treatment on the test day. In experiment 2 the animals tested in the plus-maze were left in their home cages in groups of five up to and including the test day. Drugs. Diazepam (1 mg/ml) (courtesy of Roche Products Ltd) was suspended in distilled water containing 1 drop of Tween 20 per 20 ml water, and sonicated in an ultrasonic water bath for 30 minutes prior to intraperitoneal administration. Rats allocated to chronic diazepam treatment received daily injections of 2 mg/kg. The vehicle treated animals received 2 ml/kg distilled water containing i drop of Tween 20 per 20 ml water. Zacopride (the racemic mixture, courtesy of Delalande, France) and 1-(3-chloro-phenyl)biguanide; mCPB (courtesy of MSD, UK) were dissolved in distilled water and administered intraperitoneally 30 and 15 min, respectively, prior to testing. Rats allocated to the acute control treatment groups received distilled water injections at the appropriate times before test. Apparatus. The social interaction test arena was a wooden box 60 x 60 cm with 35 cm high walls and was lit by dim light (35
radiometric lux). A camera was mounted vertically above the arena and the rats were observed from a video monitor in the adjacent room. Infra-red photocells were located in the walls of the box; those 4.5 cm above the floor recorded locomotor activity and those 12.5 cm above the floor recorded rearing of the animals. The output from both sets of photocells and the scores of the observer were entered into a computer for later analysis (see File 1980 for more details of this test). The plus-maze was made of wood and consisted of two opposite open arms - 50 cm x 10 cm and two opposite closed arms 50 x 10 x 40 cm with an open roof. The arms were connected by a central square 10 x 10 cm, thus the maze formed a "plus" shape. The maze was elevated 50 cm from the floor.
Procedure. In experiment 1,200 animals were randomly allocated between the two chronic treatment groups, such that 100 rats received an IP injection of vehicle on each of 21 consecutive days, and 100 rats received 21 daily injections of 2 mg/kg IP diazepam. On day 20 the rats were familiarised with the social interaction arena in the undrugged state, with their partners, for 4.5 min and then injected according to their normal chronic treatment. The following day the animals were placed alone in the box (again in the undrugged state) for 7.5 rain, and injected immediately after removal from the box. This was in order to test all animals in the low light, familiar test condition, which is the most sensitive to anxiogenic effects. Twenty-four hours after the 21 st injection, animals of both chronic treatment groups were randomly assigned to either control or zacopride (0.1, 0.01, 0,001 or 0.0001 mg/kg) groups and tested for 4.5 min in social interaction. The time spent in active social interaction was scored by an observer blind to the drug treatment. At the end of each trial the arena was thoroughly wiped. In experiment 2, 44 altimals were treated for 21 days with either vehicle or diazepam and on day 22 were injected with vehicle, 1 or 10 mg/kg mCPB 15 min prior to being placed singly in the same arena as was used for the social interaction test. This was in order to obtain automated measures of locomotor activity and rearing. After 5 min the animals were immediately placed in the plus-maze facing a closed arm and observed for a further 5 rain. During this time the number of open and closed arm entries and the time spent on the open and closed arms were recorded and entered into an IBM computer for later analysis. Statistics. Data were initially analysed by two factor analysis of variance with chronic treatment as one factor and acute treatment as the other factor. A significant acute x chronic treatment interaction would indicate that acute drug treatment was producing different effects depending on whether the animals had previously received chronic vehicle or diazepam. Post-hoc Duncan tests were used for comparisons of the effects of individual doses with the relevant control group.
Results It c a n be seen f r o m Fig. 1 that the rats w i t h d r a w n f r o m d i a z e p a m a n d given n o acute d r u g t r e a t m e n t h a d significantly lower social i n t e r a c t i o n scores t h a n the c o n t r o l treated animals, i n d i c a t i n g a n a n x i o g e n i c w i t h d r a w a l response. There was a significant c h r o n i c x acute treatm e n t i n t e r a c t i o n [F(4,88)= 2.5, P = 0.05], i n d i c a t i n g that there was a different response to zacopride d e p e n d i n g o n the previous c h r o n i c t r e a t m e n t . I n the c o n t r o l - t r e a t e d rats, zacopride tended to reduce the time spent in social i n t e r a c t i o n , whereas in the d i a z e p a m - w i t h d r a w n rats the trend was to increase social i n t e r a c t i o n , see Fig. 1. O n post-hoc tests, the reversal o f the a n x i o g e n i c w i t h d r a w a l response reached significance for rats treated with zacopride (0.001 mg/kg), a n d the a n x i o g e n i c effect in c o n t r o l -
335 140-
120-
DIAZEPAM WITHDRAWAL
CONTROL
(0
u~ 1 2 0 Z
o
~S
